Denis J. Moss

HLA-B27–Restricted Antigen Presentation in the Absence of Tapasin Reveals Polymorphism in Mechanisms of HLA Class I Peptide Loading

Tapasin is a resident ER protein believed to be critical for antigen presentation by HLA class I molecules. We demonstrate that allelic variation in MHC class I molecules influences their dependence on tapasin for peptide loading and antigen presentation. HLA-B*2705 molecules achieve high levels of surface expression and present specific viral peptides in the absence of tapasin. In contrast, HLA-B*4402 molecules are highly dependent upon human tapasin for these functions, while HLA-B8 molecules are intermediate in this regard. Significantly, HLA-B*2705 like HLA-B*4402, requires tapasin to associate efficiently with TAP (transporters associated with antigen processing). The unusual ability of HLA-B*2705 to form peptide complexes without associating with TAP or tapasin confers flexibility in the repertoire of peptides presented by this molecule. We speculate that these properties might contribute to the role of HLA-B27 in conferring susceptibility to inflammatory spondyloarthropathies.

New Type B Isolates of Epstein--Barr Virus from Burkitt's Lymphoma and from Normal Individuals in Endemic Areas

All Epstein-Barr virus (EBV) isolates can be classified as type A or type B depending upon the identity of their EBV nuclear antigen (EBNA) 2 protein. The great majority of isolates examined to date encode an EBNA 2A protein like that of the reference type A strain B95-8. Type B virus strains, encoding an antigenically distinct EBNA 2B protein, have as yet only been rescued from rare Burkitts lymphoma (BL) cell lines of African origin (Jijoye, AG876). Our recent finding that type B isolates are less efficient than type A in in vitro transformation assays prompted us to determine (i) the relative contribution the two types of virus make to the incidence of BL in endemic areas of Africa (Kenya) and New Guinea and (ii) the relative incidence of infection with these two types in the normal population in these same areas. On the first point, EBNA 2 gene typing using specific DNA probes showed that four of ten recently established Kenyan BL cell lines and two of four BL cell lines from New Guinea carried type B virus isolates. To address the second point, spontaneous lymphoblastoid cell lines were established from the blood of normal virus carriers and typed for EBNA 2 at the protein level; a significant proportion (greater than 20%) of the normal population in both the above BL-endemic areas were infected with type B isolates. This is the first indication of the widespread nature of type B virus infection in any community and the first isolation of such viruses from a non-BL source. The reproducible size of the EBNA 2B protein encoded by all type B isolates irrespective of their geographical origin, and of the EBNA 1 protein encoded by all type B isolates from one area, contrasted markedly with the extreme variability in the size both of EBNA 2A and of EBNA 1 seen generally among type A isolates. This suggests that the number of type B virus strains in existence worldwide could be quite limited. Most importantly, the data suggest that type B viruses, despite their relatively poor performance in in vitro transformation assays, can contribute at least as efficiently as can type A viruses to the pathogenesis of BL.

The regulatory T cell-associated transcription factor FoxP3 is expressed by tumor cells.

FoxP3 is a member of the forkhead family of transcription factors critically involved in the development and function of CD25(+) regulatory T cells (Treg). Until recently, FoxP3 expression was thought to be restricted to the T-cell lineage. However, using immunohistochemistry and flow cytometric analysis of human melanoma tissue, we detected FoxP3 expression not only in the tumor infiltrating Treg but also in the melanoma cells themselves. FoxP3 is also widely expressed by established human melanoma cell lines (as determined by flow cytometry, PCR, and Western blot), as well as cell lines derived from other solid tumors. Normal B cells do not express FoxP3; however, expression could be induced after transformation with EBV in vitro and in vivo, suggesting that malignant transformation of healthy cells can induce FoxP3. In addition, a FOXP3 mRNA variant lacking exons 3 and 4 was identified in tumor cell lines but was absent from Treg. Interestingly, this alternative splicing event introduces a translation frame-shift that is predicted to encode a novel protein. Together, our results show that FoxP3, a key regulator of immune suppression, is not only expressed by Treg but also by melanoma cells, EBV-transformed B cells, and a wide variety of tumor cell lines.

Endogenous Presentation of CD8 T Cell Epitopes from Epstein-Barr Virus-encoded Nuclear Antigen 1

Epstein-Barr virus (EBV)–encoded nuclear antigen (EBNA)1 is thought to escape cytotoxic T lymphocyte (CTL) recognition through either self-inhibition of synthesis or by blockade of proteasomal degradation by the glycine-alanine repeat (GAr) domain. Here we show that EBNA1 has a remarkably varied cell type–dependent stability. However, these different degradation rates do not correspond to the level of major histocompatibility complex class I–restricted presentation of EBNA1 epitopes. In spite of the highly stable expression of EBNA1 in B cells, CTL epitopes derived from this protein are efficiently processed and presented to CD8+ T cells. Furthermore, we show that EBV-infected B cells can readily activate EBNA1-specific memory T cell responses from healthy virus carriers. Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent. We also show that the endogenous presentation of these epitopes is dependent on the newly synthesized protein rather than the long-lived stable EBNA1. Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8+ T cell epitopes from EBNA1.

Adoptive transfer of autologous Epstein‐Barr virus–specific cytotoxic T cells for nasopharyngeal carcinoma

Tumor cells from NPC patients are regularly and latently infected with EBV. To examine whether the virus serves as target for immune intervention of the cancer, we determined levels of EBV‐specific CTLp in peripheral blood from NPC patients, long‐term survivors of the cancer and healthy subjects. CTLp levels of test subjects varied between 3– 3,000/106 PBMCs. The plasma EBV burden increased when the CTLp level fell below 150, whereas the EBV burden of PBMCs was not correlated with CTLp level. Compared with healthy carriers, CTLp levels of patients were lower and varied over a wider range, between 3–1,500/106 PBMCs. The quantitative immune deficit was probably attributed to the tumor because, first, CTLp in survivors was restored to levels similar to those in healthy carriers after the tumor had been successfully treated. Second, the CTLp level changed as disease progressed, being lower in local disease, increased in locoregional disease and decreased again when the tumor metastasized. Based on these findings, 4 patients with advanced disease were infused with 5 × 107–3 × 108 autologous EBV CTLs. The treatment was safe and unaccompanied by inflammatory or other complications, but whether it improved tumor control could not be discerned from the large tumor bulk. Nevertheless, the treatment regularly increased CTLp levels of patients, maintained it at higher levels for protracted periods and, in 3 patients, restored host surveillance of EBV replication, reducing the plasma EBV burden. Taken together, these results provided a rationale to further explore EBV as a target of immune intervention of NPC.

Vaccine strategies against Epstein-Barr virus-associated diseases: lessons from studies on cytotoxic T-cell-mediated immune regulation.

Summary: Development of a vaccine against Epstein‐Barr virus (HBV) is constrained by the latency phenotypes adopted by different EBV‐associated diseases. Over the last few years an immense body of information on the pattern of viral gene expression in EBV‐associated diseases and the rote of cytotoxic T cells in the control of these diseases has accumulated. It would seem reasonable to suggest that emerging technologies are at a level where vaccine trials aimed at controlling infectious mononucleosis, post‐transplant lymphoproliferative disease, nasopharyngeal carcinoma and Hodgklns disease are justified. On the other hand, a more cautious approach may be required for the development of vaccines or immunotherapeutic strategies against Burkitts lymphoma.

A case report: Immune responses and clinical course of the first human use of granulocyte/macrophage-colony-stimulating-factor-transduced autologous melanoma cells for immunotherapy

Abstract The first use of granulocyte/macrophage-colony-stimulating-factor-transduced, lethally irradiated, autologous melanoma cells as a therapeutic vaccine in a patient with rapidly progressive, widely disseminated malignant melanoma resulted in the generation of a novel antitumour immune response associated with partial, albeit temporary, clinical benefit. An initially negative reaction to non-transduced, autologous melanoma cells was converted to a delayed-type hypersensitivity (DTH) reaction of increasing magnitude following successive vaccinations. While intradermal vaccine sites showed prominent dendritic cell accrual, DTH sites revealed a striking influx of eosinophils in addition to activated/memory T lymphocytes and macrophages, recalling the histology of challenge tumour cell rejection in immune mice. Cytotoxic T lymphocytes (CTL) reactive with autologous melanoma cells were detectable at high frequency after vaccination, not only in limiting-dilution analysis, but also in bulk culture without added cytokines. Clonal analysis of CTL showed a conversion from a purely CD8+ response to a high proportion of CD4+ clones following vaccination. A prominent acute-phase response manifested by a five- to tenfold increase in C-reactive protein was observed, as was a systemic eosinophilia. Vaccination resulted in the regression of axillary lymphatic metastases, stabilisation of pulmonary metastases, and a dramatic, reversible increase in cerebral oedema associated with multiple central nervous system metastases; however, lesions in the adrenal glands, pancreas and spleen proved refractory. The antitumour effects and immune response were not detectable 2 months following the last vaccination. Irradiation of the extensive cerebral metastases resulted in rapid deterioration and death of the patient.

Poxvirus CD8+ T-Cell Determinants and Cross-Reactivity in BALB/c Mice

ABSTRACT Mouse models of orthopoxvirus disease provide great promise for probing basic questions regarding host responses to this group of pathogens, which includes the causative agents of monkeypox and smallpox. However, some essential tools for their study that are taken for granted with other mouse models are not available for these viruses. Here we map and characterize the initial CD8+ T-cell determinants for poxviruses in H-2d-haplotype mice. CD8+ T cells recognizing these three determinants make up around 40% of the total responses to vaccinia virus during and after resolution of infection. We then use these determinants to test if predicted conservation across orthopoxvirus species matches experimental observation and find an unexpectedly cross-reactive variant peptide encoded by ectromelia (mousepox) virus.

Five new cytotoxic T cell epitopes identified within Epstein-Barr virus nuclear antigen 3.

Epstein-Barr virus (EBV) CD8+ cytotoxic T lymphocyte (CTL) epitopes are currently being considered for inclusion into subunit vaccines. Here we describe the characterization of five new CTL epitopes within EBV nuclear antigen 3 (EBNA3), confirming EBNA3 as a major target for CTL recognition.

Effective Treatment of Metastatic Forms of Epstein-Barr Virus–Associated Nasopharyngeal Carcinoma with a Novel Adenovirus-Based Adoptive Immunotherapy

Nasopharyngeal carcinoma (NPC) is endemic in China and Southeast Asia where it is tightly associated with infections by Epstein-Barr virus (EBV). The role of tumor-associated viral antigens in NPC renders it an appealing candidate for cellular immunotherapy. In earlier preclinical studies, a novel adenoviral vector-based vaccine termed AdE1-LMPpoly has been generated that encodes EBV nuclear antigen-1 (EBNA1) fused to multiple CD8(+) T-cell epitopes from the EBV latent membrane proteins, LMP1 and LMP2. Here, we report the findings of a formal clinical assessment of AdE1-LMPpoly as an immunotherapeutic tool for EBV-associated recurrent and metastatic NPC. From a total of 24 patients with NPC, EBV-specific T cells were successfully expanded from 16 patients with NPC (72.7%), whereas six patients with NPC (27.3%) showed minimal or no expansion of virus-specific T cells. Transient increase in the frequencies of LMP1&2- and EBNA1-specific T-cell responses was observed after adoptive transfer to be associated with grade I flu-like symptoms and malaise. The time to progression in these patients ranged from 38 to 420 days with a mean time to progression of 136 days. Compared with patients who did not receive T cells, the median overall survival increased from 220 to 523 days. Taken together, our findings show that adoptive immunotherapy with AdE1-LMPpoly vaccine is safe and well tolerated and may offer clinical benefit to patients with NPC.